Biochemical and Biophysical Research Communications
Nitric oxide regulates interactions of PMN with human brain microvessel endothelial cells
Section snippets
Materials and methods
Human brain microvessel endothelial cells. Primary cultures of human brain microvessel endothelial cells (HBMEC) were established from brains at autopsy as previously described [17]. The endothelial nature of these cells was confirmed by the positive staining for Factor VIII related antigen and binding of Ulex europeaus agglutinin. Cells were grown on fibronectin coated 96-well plates and cultured in Medium 199 with 10% horse serum. Nine- to ten-day-old confluent cultures were used. Several
Results
HBMEC were first treated with TNF-α, with or without the various donors and inhibitors for 4 or 24 h; 4 h for maximal upregulation of E-selectin and 24 h for ICAM-1. PMN were then placed on top of the monolayers and allowed to adhere for 20 min. After fixation and staining, the amount of adhesion was quantified by counting the number of PMN bound in 4 peripheral and 1 central field under the microscope. TNF-α treatment increased adhesion by threefold after 4 h and fivefold after 24 h. Adhesion was
Discussion
The blood–brain barrier (BBB), formed by the endothelial cells of the cerebral microvessels, regulates what enters the brain. Adhesion and activation of circulating leukocytes is an important aspect of the inflammatory response. The BBB normally prevents the movement of leukocytes into the brain. In acute cerebral inflammatory reactions, PMN infiltrate the brain. We have previously shown that human brain microvessel endothelial cells (HBMEC) express the adhesion molecules E-selectin and ICAM-1
Acknowledgments
This study was supported by grants from the Canadian Institutes of Health Research, the Canadian Stroke Network, and the Multiple Sclerosis Society of Canada. D. Wong was the recipient of a Multiple Sclerosis Society of Canada postdoctoral fellowship. We thank Dr. Kakuri Omari, Chris Bladen, and Dorota Kwasnicka for their expert technical assistance.
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